CN113403374A

CN113403374A - Primer and method for identifying sex of melons and application of primer and method

Info

Publication number: CN113403374A
Application number: CN202110752441.7A
Authority: CN
Inventors: 张慧君; 张旭; 马建; 惠林冲; 李虎; 郑洁
Original assignee: Huaibei Normal University
Current assignee: Huaibei Normal University
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2021-09-17

Abstract

The invention provides a primer and a method for identifying the sex of melons and application thereof, belonging to the field of molecular biology. The invention aims to solve the technical problems of complicated process, low accuracy and low speed of identifying the sex of the melons. The invention provides a primer for identifying the sex of a melon, which is used for detecting the genotype of the melon by using the method for identifying the sex of the melon and judging the sex of the melon according to the genotype. The molecular marker for high-throughput detection is designed based on the G/G gene sequence for melon female floral development, and is applied to the transformation of melon female line or male line genes, so that the time and labor cost can be greatly saved, and the breeding efficiency of molecular marker-assisted selection can be improved.

Description

Primer and method for identifying sex of melons and application of primer and method

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a primer and a method for identifying the sex of melons and application of the primer and the method.

Background

Melon (2n ═ 24) (Cucumis melo L.) belongs to the Cucurbitaceae family (Cucurbitaceae) Cucumis genus (Cucumis) sprawling herbaceous plant, one of the edible fruits. The melon has unique quality and flavor, and is one of the best fruits after meals. With the development of economy and the improvement of the living standard of people, the demand of melons at home and abroad is increasing day by day. Most melon varieties are of the male hologynic type (male flowers, hologynic), i.e. the plants have unisexual male flowers and amphoteric flowers, among which the ones that can give rise to the fruit are amphoteric. The amphoteric flowers have stamens and also have pistils, and in order to obtain high-purity first-filial generation seeds, the stamens in the amphoteric flowers must be manually removed, and the artificial pollination is carried out by using the pollen of other excellent varieties, but the pollination process consumes a large amount of manpower and material resources and has great influence on the quality and yield of the melons. The melon is one of important economic crops with high yield and high efficiency, and the breeding by utilizing a female line is one of main breeding targets of seeds for protected cultivation. In the seed production process, the process of artificial pollination is saved, the high yield and the high quality can be realized, and the cost can be saved. Therefore, the research on the sex inheritance rule and the generation mechanism of the melon has important significance for guiding the melon breeding practice. The prior art has low detection efficiency and low accuracy.

In view of flowering and seed production characteristics of melon plants, at present, the labor cost of breeding and seed reproduction is rising, and how to ensure the purity of F1 generation hybrid production on the premise of reducing labor cost is always a hotspot and a pursuit target of industry attention. The defects of the prior art are as follows: in 1988, several years after the invention of the PCR technology, the allele specific Amplification (ASP)/Amplification hindered mutation system (ARMS) technology appeared. The technical principle is simple, namely, the difference of the last 1-4 bases, especially the last 1 base, of the 3' of the primer is utilized to distinguish and specifically amplify the specific allele DNA fragment. The natural DNA polymerase does not select for the last 3' bases for renaturation/mismatch and early application of this technique requires additional means to ensure selectivity. For example, artificially introducing new mismatch at the end of a primer or adjusting the annealing temperature of PCR, but these approaches are relatively complicated and have stability problems, which always plague the simplest SNP typing technique of this principle, and thus the application is not extensive.

Disclosure of Invention

The invention aims to provide a molecular marker for accurately and quickly identifying the sex of melons, and solves the technical problems of complex process, low accuracy and low speed of identifying the sex of melons in the prior art.

The invention provides a primer for identifying the sex of melons, wherein the nucleotide sequence of the primer is as follows: (a) SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3; (b) SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.

the invention also provides a kit for identifying the sex of the melons, which comprises the following primers in the group (a) or the group (b): (a) SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3; (b) SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.

further defined, the kit preferably comprises: SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11.

the invention also provides a method for determining the genotype of the melon, which comprises the following specific steps:

(1) extracting DNA of the muskmelon to be detected;

(2) performing PARMS PCR reaction by using the primer of claim 1 to obtain a typing map, wherein if the color on the typing map is FAM probe, the genotype of the melon to be detected is gg type; if the color on the typing map is the HEX probe, the genotype of the melon to be detected is GG type; if the color on the typing map is blue, the genotype of the melon to be detected is the Gg type;

(3) using SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11, carrying out PARMS PCR reaction to obtain a typing map, wherein if the color on the typing map is the color of the FAM probe, the genotype of the melon to be detected is AA type; if the color on the typing map is the HEX probe, the genotype of the melon to be detected is aa type; and if the color on the typing map is green, the genotype of the melon to be detected is Aa.

The PARMS PCR reaction system in the step (2) is as follows:

5 mu L of 2 xPARMS, DNA of the melon to be detected, 0.15 mu L of each of the primer (a) or the primer (b) and ultrapure water till 10 mu L; the group (a) of primers is SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, the primer in the group (b) is SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.

further limited, the PARMS PCR reaction system in step (3) is:

5 μ L of 2 XPAMS, DNA of the melon to be detected, SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11 to 10. mu.L each of 0.15. mu.L and ultrapure water.

Further defined, the PARMS PCR reaction conditions are:

the invention also provides a method for identifying the sex of the melon, which is to identify the genotype of the melon to be detected with CmWIP1 by using the primer and then to identify the sex of the melon with the primer of SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11, identifying the genotype of CmACS7 of the melon to be detected, determining the sex of the melon according to the genotype of CmWIP 1and the genotype of CmACS7, and if the genotype of CmACS7 is AA and the genotype of CmWIP1 is gg, determining that the melon to be detected is a full-female plant; if the genotype is aaGG, the melon to be detected is an amphoteric flower plant, if the genotype is aaGG, the melon to be detected is a male holomorph plant, and if the genotype is CmACS7 is AA, and the genotype is CmWIP1 is GG, the melon to be detected is a hermaphrodite isoflor plant.

The invention also provides application of the molecular marker or the kit in cultivation of a female or male sex system of the melon.

The method for identifying the sex of the melon or the method for determining the genotype of the melon is applied to directional sex breeding of the melon.

Has the advantages that: the invention designs a primer according to the key mutation site of a target gene based on the PARMS technology, and carries out SNP (single nucleotide polymorphism) typing on the target gene by utilizing the specific matching of the base at the tail end of the primer. The high-flux molecular marker system based on the PARMS technology has full-automatic operation process and reduces human errors; the method has high analysis flux, is compatible with 96, 384 and 1536 multi-well plates, can complete 20 to 500000 SNP genotyping every day, and is very suitable for simultaneously detecting a large number of samples. The molecular marker for high-throughput detection is designed based on the female G/G gene sequence of the melon, and is applied to the transformation of female genes of the melon, so that the time and labor cost can be greatly saved, the breeding efficiency of molecular marker assisted selection is improved, and the transformation of female characters of the melon to excellent backbone inbred lines is accelerated.

Drawings

FIG. 1 is a plot of the CmACS7 gene of Cucumis melo, wherein the abscissa is FAM fluorescence and the ordinate is HEX fluorescence;

FIG. 2 is a plot of the CmWIP1 gene of Cucumis melo, wherein the abscissa is FAM fluorescence and the ordinate is HEX fluorescence;

FIG. 3 is a graphical representation of the typing of the CmWIP1 gene of melon with FAM fluorescence on the abscissa and HEX fluorescence on the ordinate.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

gg genotype materials: WI998, Bulgarie14, Paul, Gynadou, Gynadou; GG genotype of material: p1161375, PI124112, Vedrantais, materials of the above known genotypes are described in A transposon-induced genetic changes to a ex determination in the mean.

The muskmelon full female line WI998 (without male flower) and the isogamic male and female strain 3-2-2 (with male flower) are recorded in strong way, Ma hong, Liu hong Yu, etc. the genetic analysis and the preliminary location of the muskmelon male flower differentiation gene are controlled [ J ] the university report of northeast agriculture 2010(07):51-56.

Example 1 primer design for sex determination of melon

Firstly, the GenBank accession number of the melon sex gene related to the invention is GQ870274.1and GQ870275.1 which is CmWIP1 gene, and DNA is extracted from a melon sample;

putting the leaf with the length and the width of about 1cm into a deep-hole plate (96 holes and 1.2ml), adding 100 mu L of 0.3M sodium hydroxide, grinding the leaf with 50HZ for 1min (Shanghai Jingxin tissue grinder), centrifuging at 3000rpm after grinding for 1min, boiling in a water bath for 1min, adding 400 mu L of 0.2M Tris-HCl, uniformly mixing, boiling in the water bath for 1min again, centrifuging at 3000rpm for 1min after the water bath is finished, and taking the supernatant to dilute by 10-30 times to prepare a template for subsequent PCR amplification.

Secondly, designing a specific primer: based on the CmWIP1 gene of Cucumis melo, primers were designed to the DNA sequences of the G and G genes to amplify specific fragments of G and G genotype material, and the primers designed are shown in Table 1.

PARMS PCR reactions were performed using 5 primers (primers shown in Table 1), wherein 2 fluorescent universal primers were included in the PARMS 2X Master Mix. The other 3 are labeled specific primers, one is a specific primer (Locus specific primer) of the labeled Locus, and the other 2 are SNP Allele specific primers (Allle specific primer). The 5' of the 2 primers is respectively added with a specific universal adaptor sequence of 21 bases for matched amplification with a fluorescent universal primer, and the adaptor sequence matched with FAM fluorescence is shown asGAAGGTGACCAAGTTCATGCT(SEQ ID NO: 7) linker sequence matched to HEX fluorescence isGAAGGTCGGAGTCAACGGATT(SEQ ID NO：8)。

TABLE 1 primer sequences

Specificity and accuracy of primers: according to the known genotype of the material (GG genotype: WI998, Bulgarie14, Paul, Gynadou, Gynadou; GG genotype: P1161375, PI124112, Vedrantais) through (a) SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3; and (b) SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, carrying out PARMS PCR reaction, wherein the result is shown in figure 2 and figure 3, and if the color on the typing map is blue, the genotype of the melon to be detected is gg type; if the color on the typing map is red, the genotype of the melon to be detected is GG type; if the color on the typing map is green, the genotype of the melon to be detected is Gg type; FIG. 2 is a graph utilizing SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 sequence PARMS PCR reaction to obtain typing map; FIG. 3 is a graph utilizing SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: the 6 sequences were scored.

According to the materials of known genotype (WI998, Bulgarie14, Paul, Gynaadou; P1161375, PI124112, Vedrantais), the results are shown in FIG. 1 using the sequences shown in SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11, carrying out PARMS PCR reaction to obtain a typing map, wherein if the color on the typing map is blue, the genotype of the melon to be detected is AA type; if the color on the typing map is red, the genotype of the melon to be detected is aa type; and if the color on the typing map is green, the genotype of the melon to be detected is Aa. (GenBank accession numbers of the GmACS7 gene are EU791279.1 and EU 791280.1).

The results obtained with the above primers were also identical to those obtained with the PARMS PCR reaction on melon varieties of known genotypes as shown in table 2.

TABLE 2 genotype results analysis

Example 2 kit for sex determination of melon

The kit comprises the following components: (a) SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3; and (b) SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, PARMS Mix. The use method of the kit comprises the following steps: and (3) carrying out PARMS reaction by using the DNA of a sample to be detected as a template and the kit, analyzing the genotype of the melon, and judging the sex of the melon according to the genotype of the melon.

Example 3 method for sexing melon

Firstly, sample selection: selecting a muskmelon Boyang No. 9 183 strain with known sex;

secondly, sample processing: placing leaves of melon with length and width of about 1cm into a deep-well plate (96 holes with 1.2ml), adding 100 mu L of 0.3M sodium hydroxide, grinding at 50HZ for 1min (Shanghai Jingxin tissue grinder), centrifuging at 3000rpm for 1min after grinding, boiling in a water bath for 1min, adding 400 mu L of 0.2M Tris-HCl, mixing uniformly, boiling in a water bath for 1min again, centrifuging at 3000rpm for 1min after the water bath is finished, and taking supernatant to dilute by 10-30 times to prepare a template for subsequent PCR amplification.

PARMS system formulations are shown in tables 3 and 4:

TABLE 3 PARMS systems

TABLE 4 PARMS systems

The minimum reaction systems of the 96-well PCR plate and the 384-well plate are 10ul and 5ul, respectively, and are shown in Table 5. PARMS is compatible with 0.8 ul-2.0 ul small-line platform. For long-term storage, the PARMS is placed at the temperature of-20 ℃ and stored at the temperature of 4 ℃ for 2 weeks, and the PARMS Mix freeze-thaw duration is more than 3 times.

TABLE 5 PARMS PCR reaction size

The PARMS thermocycling program is shown in table 6:

TABLE 6 PARMS thermocycling program

Third, genotyping result analysis

After the PARMS PCR is finished, reading a fluorescent signal by using a TECAN infiinite M1000 microplate reader according to the group (a) of primers and the group (b) of primers, analyzing and converting the fluorescent signal by using online software snpdecoder to obtain a clear and visual parting diagram, outputting a genotype result according to different colors, and then using a primer sequence shown in SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11 the PARMS PCR reaction was performed to obtain a typing chart, and the results are shown in Table 7, and the results of the detection match the actual sex of melon. The identification result is shown in table 7, if the genotype of CmACS7 is AA and the genotype of CmWIP1 is gg, the melon to be detected is a full female plant; if the genotype is aaGG, the melon to be detected is an amphoteric flower plant, if the genotype is aaGG, the melon to be detected is a male holomorph plant, and if the genotype is CmACS7 is AA, and the genotype is CmWIP1 is GG, the melon to be detected is a hermaphrodite isoflor plant.

Table 7 identification results

Example 4 application of primers for identifying melon sex in melon breeding

Selecting a melon variety with unknown sex to carry out PARMS PCR reaction to obtain the genotype of the melon, identifying the sex of the parents, and then hybridizing to obtain the melon with the directional sex.

SEQUENCE LISTING

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Claims

1. A primer for identifying the sex of melon, wherein the nucleotide sequence of the primer is the primer in the (a) group or the (b) group: (a) SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3; (b) SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.

2. a kit for identifying the sex of melon, which comprises the primers in group (a) or group (b):

(a) SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3;

(b) SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.

3. the kit of claim 2, wherein the kit further comprises: SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11.

4. a method for determining the genotype of a melon is characterized by comprising the following specific steps:

(1) extracting DNA of the muskmelon to be detected;

5. The method of claim 4, wherein the PARMS PCR reaction system of step (2) is: 5 mu L of 2 xPARMS, DNA of the melon to be detected, 0.15 mu L of each of the primer (a) or the primer (b) and ultrapure water till 10 mu L; the group (a) of primers is SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, the primer in the group (b) is SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.

6. the method of claim 4, wherein the PARMS PCR reaction system of step (3) is: 5 μ L of 2 XPAMS, DNA of the melon to be detected, SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11 to 10. mu.L each of 0.15. mu.L and ultrapure water.

7. The method of claim 4, wherein the PARMS PCR reaction conditions are:

8. a method for identifying the sex of melon by using the primers of claim 1 to identify the genotype of CmWIP1 of melon to be tested and then using the primers of SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11, identifying the genotype of CmACS7 of the melon to be detected, determining the sex of the melon according to the genotype of CmWIP 1and the genotype of CmACS7, and if the genotype of CmACS7 is AA and the genotype of CmWIP1 is gg, determining that the melon to be detected is a full-female plant; if the genotype is aaGG, the melon to be detected is an amphoteric flower plant, if the genotype is aaGG, the melon to be detected is a male holomorph plant, and if the genotype is CmACS7 is AA, and the genotype is CmWIP1 is GG, the melon to be detected is a hermaphrodite isoflor plant.

9. Use of the molecular marker of claim 1 or the kit of claim 2 or 3 for breeding a melon female or male sex system.

10. Use of the method of any one of claims 4 to 7 or the method of claim 8 for directional sex breeding of melons.